Throughout this application various publications are referred to in square brackets. Full citations for these references may be found at the end of the specification. The disclosures of these publications, and all patents, patent application publications and books referred to herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Antibodies are superior therapeutic reagents because of their wide range of biological functions, naturally evolved sustainable half-life and comparatively good safety. The effectiveness and success of an antibody-mediated therapy rely heavily on the specificity and affinity of the antibody against cognate antigens. Thus, obtaining antibodies with clinical application potential for designated therapeutic target represents a major hurdle in current bio-pharmacological industry.
Platforms to diversify antibodies in vitro offer a valuable alternative to traditional immunization based antibody production approach. They offer the possibility to overcome the hurdle encountered by the host immune system during the immunization process such as low immunogenicity, fast antigen degradation, immune dominancy etc. Currently, phage display and E. coli based gene expression library are the two main in vitro methods for antibody diversification. However, these processes do not provide post-translational modifications that are important for antibody binding and do not take advantage of antibody scaffolds that are evolutionarily evolved to provide high specific antibody-antigen interactions.
Vertebrate cells that undergo constitutive somatic hypermutation (SHM) at antibody gene loci have been shown to have the potential of generating de novo antibody against the model antigen streptavidin. However, the mutation frequency of those cells is 1-2 log scale lower than the somatic hypermutation (SHM) process that occurs in normal germinal center B cells in vivo. Therefore, commercial application of eukaryotic cell based antibody diversification process using physiological affinity maturation mechanism such as AID mediated mutation and error-prone DNA damage repair to generate a de novo antibody from an immunoglobulin gene that does not encode an antibody and that recognizes a predetermined antigen has been difficult, and still requires an the increase of in vitro SHM rate.
Over expression of the B cell endogenous genomic mutating factor activation induced deaminase (AID) in human Ramos cell lines can increase SHM rate at immunoglobulin gene (Ig) loci (FIG. 1) less than 10 fold. However, due to the genotoxicity of AID, further improvement of mutation rate just based on the modulation of the level or the activity of AID and its related molecules has been difficult to achieve. Some microbial products, such as LPS, have been suggested to increase AID level in B cells but there has been no evidence that these reagents can increase SHM rate in B cells in vitro. Thus, alternative approaches to enhance SHM at Ig loci are urgently needed to help establish eukaryotic cell-based antibody diversification platforms that offer a clear advantage over techniques currently available in the market.
The present invention addresses the need for alternative approaches to enhance the antibody diversification process through SHM at Ig loci, especially in vitro.